Apolipoprotein A-I (apo A-I), which binds discoidal and spherical lipid surfaces via a series of 11- and 22-mer amphipathic helices, is the major protein constituent of high density lipoprotein (HDL). Apo A-I has antiatherogenic properties, which include reverse cholesterol transport and inhibition of LDL oxidation. The molecular mechanisms governing these and other apo A-I functions are largely unknown. Previous work demonstrating proteolytic susceptibility near residue 42 in lipid-bound but not lipid-free apo A-I stimulated the design of a deletion mutant, delta 1-43 apo A-I. Removal of residues 1-43 resulted in conversion of the lipidfree protein structure to the lipid-bound conformation, even in the absence of lipid, suggesting that this region acts as a conformational switch between lipid-free and lipid-bound protein conformations. The crystal structure of delta 1-43 apo A-I (the only crystal structure of apo A-I to date) provided critical information concerning the disposition of amphipathic helices on lipid surfaces. However, it provides no information concerning the structure of residues 1-43, which have been proposed to form a globular domain. Knowledge of the structure in this region of the protein is critical for understanding apo A-I's anti-atherogenic properties as well as for understanding mechanisms governing the propensity for amino-terminal mutants of apo A-I to form amyloids. Our research aims to fill the gap in knowledge of the 1-43 region through two Specific Aims: we will delineate the portion of 1-43 needed to maintain the protein in its lipid-free state by analyzing the structures of proteins with smaller deletions within the 1-43 region (Specific Aim 1), and evaluate the conservation of the conformational switch properties of the 1-43 region by comparison of full-length and delta 1-43 analogs of zebrafish apo A-I (Specific Aim 2). The structures and lipid-binding properties of the proposed mutants will be evaluated using spectroscopic and physico-chemical techniques that have been shown to distinguish the normal lipid-free structure from that of the delta 1-43 protein including circular dichroism and fluorescence spectroscopy, limited proteolysis, reconstituted HDL formation, and disassembly of multilamellar vesicles. [unreadable] [unreadable]